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1Hematology/Oncology, Dept of Medicine, University of Illinois at Chicago, Chicago, IL2Department of Biology, Loyola University Chicago, Chicago, IL3Section of Hematology/Oncology, Department of Medicine, University of Illinois at Chicago, Chicago, IL

Heme, a complex of
iron and protoporphyrin IX, is ubiquitous in aerobic cells, serving as the
prosthetic group for essential hemoproteins. However, an excess of
intracellular “free” heme is toxic to cells, promoting lipid peroxidation,
membrane injury, reactive oxygen species (ROS) production, and ultimately
apoptosis, necessitating tight regulation. FLVCR is a human erythroid
progenitor cell membrane transporter that exports heme from the cytosol,
thereby protecting cells from heme toxicity (Quigley et al. Cell, 2004). It is
also highly expressed at sites of heme trafficking including the liver, gut,
kidney and erythrophagocytic macrophages. In accordance with its function and
expression pattern, our knockout mouse model demonstrates that FLVCR is important
both for erythropoiesis and for systemic iron homeostasis (Keel et al. Science
2008). Female mosquitoes ingest ~10 times their weight in blood in a single
meal, thus large quantities of heme are released into the midgut lumen, a
source of toxic ROS. Notably, hematophagy also represents a major bottleneck in
the Plasmodium lifecycle. Gametes egressing from disintegrating RBCs are
fertilized within the midgut lumen, forming small numbers of ookinetes that are
susceptible to ROS as they traverse the mosquito midgut epithelial cells (to
develop into oocysts). Indeed, numerous studies demonstrate that mosquitoes
with increased midgut oxidative stress are resistant to Plasmodium
transmission. We hypothesize that dysregulation of anopheline FLVCR–mediated
heme export by the mosquito midgut epithelium will increase epithelial cell
oxidative stress and impact Plasmodium transmission at its weakest
point.

Using genomic approaches, we isolated anopheline
orthologs of FLVCR from the prevalent malaria transmission vectors, Anopheles
gambiae and A. stephensi. The AgFLVCR cDNA predicts a 51kDa
protein of 474 aa; the sequence of AsFLVCR (474 aa) is 97% identical. Like
FLVCR, both genes are predicted to encode Major Facilitator Superfamily
transporter proteins containing 12 TM domains, with the N– and C–termini in the
cytosol. Overexpression of AgFLVCR cDNA in (rat) NRK cells results in an
increase in export of ZnMP (a fluorescent heme analog) comparable to that
observed with NRK cells overexpressing human FLVCR—a rate significantly greater
(~2–fold) than that of control NRK cells, as measured by quantitative
microscopy (n=4, p<0.001). Similar increases in heme export relative
to controls are observed when (insect) Sf9 cells are transduced with Asflvcr
or Agflvcr and ZnMP export analyzed using flow cytometry. The increase
is blocked by specific siRNA against Asflvcr or Agflvcr. As
expected, incubation with exogenous heme (20 µM) reduces viability of NRK
cells. However, overexpression of AsFLVCR in NRK cells, by reducing
intracellular ROS levels by ~ 40%, improves their viability 3–fold (n=3, p<0.001).
In studies of A. stephensi mosquitoes, we demonstrate robust
upregulation of AsFLVCR mRNA (Fig. A, left) and protein (right) at 24 h
after hematophagy, while confocal fluorescence immunolocalization studies with
an antibody (Ab) generated against an intracellular epitope of AsFLVCR show
cell membrane targeting in midgut epithelial cells following a blood meal (Fig.
B; blue, DAPI; green, AsFLVCR). Importantly, dsRNA-mediated knockdown of
AsFLVCR in mosquitoes decreases midgut Asflvcr mRNA by 85%, resulting in
a 30% reduction in protein levels post-hematophagy. A polyclonal Ab
(α-AsFLVCR) was also generated against an extracellular epitope.
Preliminary studies indicate it impairs heme export from Sf9 cells
overexpressing AsFLVCR. At present we are evaluating the effects of
α-AsFLVCR– or dsRNA–mediated blockade of AsFLVCR heme export function on Plasmodium
oocyst survival, salivary gland sporozoite development, mosquito midgut ROS
production and mosquito fecundity. If these results validate our hypothesis,
modulation of anopheline FLVCR–like proteins may serve as a means of
controlling malaria.